Leaf discs (3.8 mm in diameter) were obtained from young fully expanded but not hardened leaves and were kept in 200 µl of sterile water overnight in a 96-well plate at room temperature. The next day, the water was replaced with 100 µl of assay solution (100 µM luminol, 10 µg of horseradish peroxidase per milliliter, and either 100 nM or 10 µM flg22). Assay solution without flg22 was used for controls. Luminescence (count per second, CPS) was measured for 60 min using the LUMIstar microplate luminometer (BMG LABTECH, Cary, NC, U.S.A.). Means and standard errors for ROS production peak values were calculated based on 36 leaf discs for each treatment. Experiments were repeated twice with similar results.
Lumistar microplate luminometer
Manufactured by BMG Labtech
Sourced in Germany, United States
The LUMIstar microplate luminometer is a laboratory instrument designed for the detection and quantification of luminescent signals in microplates. It is capable of measuring various luminescence-based assays, providing precise and reliable data for research and analytical applications.
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Lab products found in correlation
Celltiter glo cell viability assay, by Promega (3 mentions)
Sigmaplot 14, by Grafiti LLC (2 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Veliparib, by Selleck Chemicals (1 mentions)
Ku55933, by Selleck Chemicals (1 mentions)
Ve 821, by Selleck Chemicals (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Celltiter glo luminescence assay, by Promega (1 mentions)
10 protocols using lumistar microplate luminometer
1
Leaf Disc Assay for ROS Production
2
ATP Quantification in Citrus Leaves
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ATP concentrations were measured using a luciferin-based ATP assay (Invitrogen, Carlsbad, CA, USA). Similar to H2O2 detection, citrus leaf discs were placed in the 96 well plate at the bottom of the well except only six leaf discs were used in this assay. The ATP detection buffer was then added into the wells as per the manufacturer’s protocol and the plate was immediately placed in the LUMIstar microplate luminometer for luminescence measurements (BMG Labtech, Ortenberg, Germany).
3
ROS Induction Assay for Xcc flg22
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A peptide corresponding to the flg22 of Xcc was ordered from GenScript (Piscataway, NJ, USA) with >90% purity using the amino sequence QRLSSGLRINSAKDDAAGLAIS. The peptide was resuspended in sterile water and used to measure induction of ROS. Six leaf disks from three newly expanded young leaves were punched out with a 4-mm-diameter cork borer. Leaf discs were floated abaxial side up in an individual well of a black 96-well plate with 200 µl of water per well at room temperature overnight and covered with plastic bags. The next day the water was removed, and ROS production was triggered with 200 nM flg22 applied together with 20 μM luminol and 1 μg per 100 μL of horseradish peroxidase. Luminescence was measured by the LUMIstar microplate luminometer (BMG Labtech, Cary, NC, USA). Each plate was measured over a period of 40 min. A total of 32 one-year-old seedlings from each variety were used. The assay was repeated twice. Controls lacking flg22 were included for each species with the control lane displayed in Figure 1 consisting of a combination of five data points from each of the species tested (30 total data points). Data were analyzed using the Optima microplate reader software (BMG Labtech).
4
Dose-dependent cell viability in chondrosarcoma
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For the dose–response relationship, chondrosarcoma cells were irradiated with 0 Gy (neg. control) and 4 Gy X-ray/proton/C-ions IR. Cell viability was determined with the CellTiter-Glo® cell viability assay (Promega Corporation, Madison, MI, USA) after 24 to 168 h and normalized to the non-IR controls. Background reference values were derived from the culture media. Absorbance was measured with a LUMIstar™ microplate luminometer (BMG Labtech GmbH, Ortenberg, Germany) (mean ± SD; n = 7, performed in biological quadruplicates). The xCELLigence RTCA-DP device (OLS, Bremen, Germany) was used to monitor cell proliferation in real-time. Cells were seeded after IR in electronic microtiter plates (E-Plate™, OLS) and measured for 120 h according to the manufacturer’s instructions. Cell density was measured in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with RTCA software (version 1.2, OLS).
5
Dose-Dependent Cytotoxicity Assay
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The dose-response curves were determined using the CellTiter 96 AQueous Luminescence Assay (Promega, Madison, MA, USA). The primary OA chondrocytes (pCH-OA) and 5 × 103 HC were seeded on white 96-well plates and either used as a control or treated with acetylshikonin, shikonin, or cyclopropylshikonin in various concentrations between 0.1 and 25 µM. After a 24 h incubation period, the viability assay was performed in accordance with the manufacturer’s protocol. Background reference values were derived from the culture media. Absorbance values were measured with the Lumistar microplate luminometer (BMG Labtech, Ortenberg, Germany). IC50 values were calculated with SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA), using the four-parameter logistic curve.
6
Bortezomib Enhances Radiosensitivity
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For the dose-response relationship, 100 spheroids each of HC and SW1353 were pretreated with the IC50 concentration of 5 nM bortezomib (Selleck Chemicals) for 24 h. Cell viability was determined with the CellTiter-Glo® 3D Cell Viability assay (Promega Corporation) at 3 and 48 h post-administration of 0 (control) to 20 Gy IR. Background reference values were derived from the culture media. Absorbance was measured with a LUMIstar™ microplate luminometer (BMG Labtech GmbH), and the mean ± SD was calculated (HC, n=3; SW1353, n=7; performed in biological quadruplicates).
7
Dose-Dependent Cell Viability and Proliferation
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For the dose–response relationship, SW-1353 cells were irradiated with 0 Gy (neg. control) and 4 Gy photon respectively proton IR. Cell viability was determined with the CellTiter-Glo® cell viability assay (Promega Corporation, Madison, MI, USA) after 24–168 h and normalized to the non-IR controls. Background reference values were derived from the culture media. Absorbance was measured with a LUMIstar™ microplate luminometer (BMG Labtech GmbH, Ortenberg, Germany) (mean ± SD; n = 7, performed in biological quadruplicates). The xCELLigence RTCA-DP device (OLS, Bremen, Germany) was used to monitor cell proliferation in real-time. Cells were seeded after IR in electronic microtiter plates (E-Plate™, OLS) and measured for five days according to the manufacturer´s instructions. Cell density was measured in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with RTCA software (version 1.2, OLS).
8
Chondrosarcoma Cell Dose-Response Assay
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For the dose–response relationship, chondrosarcoma cells were pre-treated with 2.5 µM, 10 µM, and 25 µM of the PARPi Olaparib or Veliparib, the ATMi Ku-55933, or the ATRi VE-821 (all Selleckchem, Houston, TX, USA). Afterwards, the cells were irradiated with 0 Gy (non-IR control), with a fractionated IR of 8 Gy X-ray on each of the three subsequent days, or with a total dose of 24 Gy. Cell viability was determined with the CellTiter-Glo® cell viability assay (Promega Corporation, Madison, MI, USA) and normalized to the non-IR controls. Background reference values were derived from the culture media. Absorbance was measured with a LUMIstar™ microplate luminometer (BMG Labtech GmbH, Ortenberg, Germany) (mean ± SD; n = 3, performed in biological quadruplicates).
The xCELLigence RTCA-DP device (OLS, Bremen, Germany) was used to monitor cell proliferation in real-time. Cells were seeded after IR in electronic microtiter plates (E-Plate™, OLS) and measured for 120 h according to the manufacturer’s instructions. Cell density was measured in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with RTCA software (version 1.2, OLS).
The xCELLigence RTCA-DP device (OLS, Bremen, Germany) was used to monitor cell proliferation in real-time. Cells were seeded after IR in electronic microtiter plates (E-Plate™, OLS) and measured for 120 h according to the manufacturer’s instructions. Cell density was measured in quadruplicate with a programmed signal detection every 20 min. Data acquisition and analyses were performed with RTCA software (version 1.2, OLS).
9
Citrus leaf H2O2 detection assay
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H2O2 detection was conducted as described in previous studies with the following minor modifications.36 (link),50 (link)–52 (link) Briefly, citrus leaf discs were collected from either the Las-infected citrus trees or non-infected trees using a circular 5 mm diameter cork borer. Ten different replicates were performed where each replicate, consisting of a total of nine leaf discs collected from three different symptomatic leaves of a single plant, was placed into a loading buffer consisting of 50 mm Tris-KCl (pH 7.2) with 100 μm of H2DCF-DA and fluorescence emission was immediately measured by the LUMIstar microplate luminometer (BMG Labtech, Ortenberg, Germany) at excitation wavelength, 484 nm, and emission wavelength, 525 nm.
10
Chondrosarcoma Cell Viability Assay
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5 × 103 chondrosarcoma cells were seeded on white 96-well plates and either used as control or treated with acetylshikonin, shikonin, or cyclopropylshikonin in various concentrations between 0.1 and 25 µM. The dose-response curves were determined using the CellTiter-Glo® Luminescence Assay (Promega, Madison, MA, USA) according the manufacturer´s protocol after a 24 h incubation period. Untreated culture media served as reference values for the background. The viability assay was performed in biological quadruplicates (n = 6). Absorbance values were measured with the Lumistar microplate luminometer (BMG Labtech, Ortenberg, Germany) and the corresponding IC50 values were calculated with SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) using the four-parameter logistic curve.
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